Abstract Although bone morphogenetic protein-2 (BMP-2) is believed to be the most potent osteoinductive factor available for bone regeneration, its clinical application requires supraphysiological BMP doses that increase inappropriate adipogenesis and cyst-like bone formation. Therefore, recent promising alternative strategies are toward promoting pro-osteogenic activity of BMPs while simultaneously suppressing their adverse effects. Recent studies suggest important roles of tribbles homologs proteins in development and cellular differentiation. In response to BMP stimulation, in particular, tribbles homolog 3 (Trb3) is dissociated from BMP receptor and degrades Smad ubiquitin regulatory factor 1 (Smurf1), a negative regulator of BMP receptor-regulated Smads. Moreover, Trb3 has been shown to bind and suppress peroxisome proliferator-activated receptor-? (PPAR?), a master regulator of adipogenesis. Thus, Trb3 may be a promising molecular target to enhance BMP-2 induced osteogenesis and reduce adverse adipogenesis. Upon BMP stimulation, however, BMP efficacy is greatly reduced due to the enhanced expression of natural BMP antagonists to auto-regulate endogenous BMP-2 levels. Thus, the potency of Trb3 in BMP treatments can be enhanced by inhibiting expression of BMP antagonists such as noggin. The objective of this study is to investigate whether overexpression of Trb3 and simultaneous abrogation of BMP antagonism can enhance BMP-induced osteogenesis and bone formation quality while lowering exogenous BMP requirement. The specific hypothesis is that the augmentation of Trb3 expression combined with the employment of noggin suppression can enhance bone formation by activation of Smad pathway and suppression of PPAR?, tested in the following aims. In Aim 1, we will evaluate whether Trb3 overexpression can synergistically enhance bone formation with BMP-2 in a rat critical size mandibular defect (MD). We will also apply high dose BMP-2 to induce adverse cyst-like bone formation in the MD model and test whether Trb3 overexpression can reduce the BMP-2 induced adverse outcomes and improve bone formation quality. In addition, we will determine whether the positive Trb3 effects are dependent on Smurf1 and PPAR? activities in vitro. Next in Aim 2, we will stimulate endogenous BMP signaling by downregulating noggin using an RNAi strategy and evaluate the synergistic effect of noggin suppression on Trb3-induced bone formation in the MD model. We will subsequently determine whether Trb3 expression + noggin suppression can lower total BMP-2 dose requirements or even eliminate use of exogenous BMP-2 without compromising osteogenic efficacy. Finally in Aim 3, we will further elaborate our complementary bone formation strategy (Trb3 expression + noggin suppression) toward clinical translations via use of appropriate carriers that deliver these therapeutic genes in much safer and efficient manners based on our novel non-phospholipid liposomal systems. Successful completion of these studies will identify a new strategy to improve clinical efficacy and safety of current BMP therapeutics by regulating expression of Trb3 and noggin for enhanced bone repair.